Pharmaceutical compositions and methods for producing low impurity concentrations of the same

ABSTRACT

A composition having clevidipine as an active ingredient is described. The composition includes clevidipine as an active ingredient and an amount of the impurity H168/79 that is no greater than about 1.5%, or where the ratio between clevidipine and H168/79 is equal or above 60 to 1.

This application claims priority from U.S. Provisional Application No.61/085,597 filed on Aug. 1, 2008 and U.S. Provisional Application No.61/093,772 filed on Sep. 3, 2008. The above mentioned applications areincorporated herein by reference.

FIELD OF THE INVENTION

The instant invention relates to pharmaceutical compositions, and inparticular to compositions of clevidipine having a reduced level ofimpurities, and a method of maintaining the stability of suchpharmaceutical compositions.

BACKGROUND OF THE INVENTION

Clevidipine, which is also known as Cleviprex™, is a short-acting,vascular selective calcium antagonist that has been shown to reducearterial blood pressure with a fast termination of effect due tometabolism by blood and tissue esterases. As an arterial-selectivevasodilator, clevidipine reduces peripheral vascular resistancedirectly, without dilating the venous capacitance bed.

The chemical name of clevidipine is butyroxymethyl methyl4-(2′,3′-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate(C₂₁H₂₃Cl₂NO₆). Its structure is as follows:

Clevidipine is typically formulated as a liquid emulsion suitable forintravenous administration. Lipid emulsions are widely used inparenteral nutrition use for approximately 30 years and in the recentpast have been used as drug carriers for insoluble drugs such aspropofol (Diprivan®), and diazepam. Apart from their ability to deliverinsoluble drugs, emulsions are also suitable dosage forms for drugs likeclevidipine that are susceptible to hydrolytic breakdown. Emulsions havealso been reported to prevent drugs from adhering to plasticadministration sets used during intravenous injection, and reduce localtoxicity on infusion.

As a pharmaceutical composition, it is essential that clevidipinemaintains its stability. Over the past several years, various impuritieshave been identified in compositions containing clevidipine as an activeingredient. For example, some impurities arise from the process used inmaking clevidipine, while others are due to gradual degradation of theactive ingredient. As a pharmaceutical composition, it is essential tomaintain stability and minimize the amount of impurities regardless oftheir source or the mechanism of degradation. Therefore, a need existsfor compositions of clevidipine having acceptable stability profileswith respect to their ultimate potency and impurity levels. There isalso a need for methods for maintaining the stability of compositionshaving clevidipine as an active ingredient.

SUMMARY OF THE INVENTION

The first aspect of the present invention describes a number ofimpurities which may be derived from clevidipine through a hydrolysis,decarboxylation and condensation reaction. It describes the structure ofthese impurities and methods of detecting and analyzing theseimpurities.

The second aspect of the present invention describes methods of reducingthe amount of such impurities in a pharmaceutical compositions havingclevidipine as an active ingredient.

The third aspect of the present invention describes pharmaceuticalcompositions prepared or stored using the methods described hereinwherein the level of certain impurities is minimized or reduced,

In particular, the present invention describes pharmaceuticalcomposition having clevidipine as an active ingredient, and having areduced level of one or more impurities selected from a group consistingof Substance 23, Substance 24, Substance 25 and H168/79.

More specifically, the present invention describes a pharmaceuticalcomposition having clevidipine as an active ingredient, wherein thecompositions contains equal or no more than 0.2% of an impurity on aweight-to-weight of impurity to clevidipine and the impurity is selectedfrom a group consisting of Substance 23, 24 and 25.

Even more specifically, the present invention describes a pharmaceuticalcomposition having clevidipine or any of its pharmaceutically acceptablesalt forms, as the active ingredient, wherein the composition containsequal or no more than 0.2% on a weight-to-weight of impurity toclevidipine for each of the purities, Substance 23, 24 and 25.

The present invention includes compositions having clevidipine, as anactive ingredient, wherein the composition contain a reduced level of anamount of the impurity H168/79 that is no greater than about 1.5%weight-to-weight of impurity to clevidipine basis, or where the ratio ofthe area under the chromatographic curve between clevidipine and H168/79is equal or greater than 60 to 0.9.

The present invention also includes compositions having clevidipine orany of its pharmaceutical acceptable salt forms, as an activeingredient, wherein the compositions contain a reduced level of anamount of the impurities H168/79, Substance 23, Substance 24, andSubstance 25 that the level of H168/79 is no greater than about 1.5% ona weight-to-weight of impurity to clevidipine or where the ratio of thearea under the chromatographic curve between clevidipine and each ofSubstance 23, Substance 24, and/or Substance 25 is equal or greater than500 to 1, and the ratio the area under the chromatographic curve betweenclevidipine and H168/79 is equal or greater than 60 to 0.9.

The present invention also describes a method of manufacturingcompositions having clevidipine as an active ingredient, and an amountof the impurity H168/79 that is no greater than about 1.0%weight-to-weight of impurity to clevidipine, or where the ratio betweenclevidipine and H168/79 is equal or greater than 100 to 1.

The fourth aspect of the present invention is a method of treating oralleviating a disease or condition in a subject in need thereof,comprising administering to the subject an effective amount of apharmaceutical composition having clevidipine or any of itspharmaceutical acceptable salt forms as the active ingredient, whereinthe level of impurities is reduced or minimized to no more than 0.2%weight-to-weight of impurity to clevidipine for any of Substance 23,Substance 24, and Substance 25, and no more than 1.5% for H168/79 basedon a weight-to-weight of impurity to clevidipine. As used herein thedisease or condition refers to any disease or condition which may betreated using a selective calcium channel block, such as clevidipine.Examples of such disease or condition include, without limitation,hypertension, such as primary hypertension, secondary hypertension,acute hypertension, chronic hypertension, high blood

Pressure, chest pain (angina), migraine, brain aneurysm complications,irregular heartbeats (arrhythmia) and Raynaud's disease.

DESCRIPTION OF THE FIGURES

Understanding of the present invention will be facilitated byconsideration of the following detailed description of the embodimentsof the present invention taken in conjunction with the accompanyingdrawing, in which like numerals refer to like parts and in which:

FIG. 1 illustrates a proposed degradation pathway of clevidipine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is to be understood that the figures and descriptions of the presentinvention have been simplified to illustrate elements that are relevantfor a clear understanding of the present invention, while eliminating,for the purpose of clarity, many other elements found in typicalpharmaceutical compositions and methods of stabilization. Those ofordinary skill in the art will recognize that other elements and/orsteps are desirable and/or required in implementing the presentinvention. However, because such elements and steps are well known inthe art, and because they do not facilitate a better understanding ofthe present invention, a discussion of such elements and steps is notprovided herein. The disclosure herein is directed to all suchvariations and modifications to such elements and methods known to thoseskilled in the art. Furthermore, the embodiments identified andillustrated herein are for exemplary purposes only, and are not meant tobe exclusive or limited in their description of the present invention.

As mentioned previously, clevidipine is a fast acting dihydropyridinecalcium channel blocking agent developed for the treatment of variousconditions, such as hypertension, including primary hypertension,secondary hypertension, acute hypertension, chronic hypertension andperioperative hypertension in cardiac surgery, high blood pressure,chest pain (angina), migraines, brain aneurysm complications, irregularheartbeats (arrhythmia) and Raynaud's disease. As an arterial-selectivevasodilator, clevidipine reduces peripheral vascular resistancedirectly, without dilating the venous capacitance bed. The end effectcan be a reduction in systolic blood pressure. More detailed informationon short-acting dihydropyridines and their clinical indications can befound in U.S. Pat. No. 5,856,346, the entire disclosure of which isincorporated by reference herein as if set forth in its entirety.

As used herein, the term “clevidipine” shall mean and include allvarieties of forms of clevidipine. Unless otherwise specified, examplesof such forms include all pharmaceutically acceptably salts, esters,isomers, stereo isomers, crystalline and amorphous forms.

As used herein, the term “pharmaceutically acceptable salt” shall referto salts prepared from pharmaceutically acceptable non-toxic bases oracids including inorganic or organic bases and inorganic or organicacids. Examples of salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc, and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium, and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, and basic ion exchange resins, such as arginine, betaine,caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine, and the like.

Clevidipine is manufactured by reaction of4-(2′,3′-dichlorophenyl)-1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3-pyridinecarboxylicacid with chloromethyl butyrate to obtain clevidipine. This reaction canbe done optionally in the presence of a corresponding hydrogencarbonate, such as KHCO₃, in refluxing acetonitrile. Inorganic salts canbe removed by filtration and the product is crystallized by the additionof isopropanol and water with subsequent cooling. It can also becrystallized by exchanging solvent from acetonitrile to a mixture ofalcohol, such as ethanol or isopropanol, and water with repeatedevaporations. In the further purification of the product the crystalsare washed with a mixture of water and ethanol or isopropanol. Theproduct can be dissolved in refluxing isopropanol, crystallized bycooling, isolated by filtration and finally washed with a water andisopropanol mixture. A more detailed description of the manufacturingprocess of clevidipine can be found in U.S. Pat. No. 6,350,877, theentire disclosure of which is incorporated by reference herein as if setforth in its entirety.

Clevidipine is practically insoluble in water, and thus is typicallyformulated as a liquid emulsion suitable for intravenous administration.Typically, each mL may contain 0.5 mg clevidipine in approximately 20%soybean oil emulsion for intravenous administration. Other ingredientsmay include glycerin, water, purified egg yolk phospholipids, and sodiumhydroxide to adjust pH.

Emulsions offer much better solubility, less side effects of the vehicleand better stability than conventional solutions. Oil-in-water emulsionsalso prevent the compound from adherence to plastic infusion sets thatare to be used when administering the compound. These emulsions providea fast release and decay, and offer much better in vivo solubilityproperties, fewer side effects of the vehicle and better stability thanconventional solutions. Further information regarding the formulation ofclevidipine can be found in U.S. Pat. No. 5,739,152, the entiredisclosure of which is incorporated by reference herein as if set forthin its entirety.

It was previously unknown that compositions having clevidipine as anactive ingredient are heat intolerant and sensitive to water content.However, based on this discovery that such adverse conditions can giverise to an unacceptable level of impurities from pharmaceuticalcompositions having clevidipine as an active ingredient, the presentinvention provides compositions that include clevidipine and minimizedimpurity levels, along with methods of manufacturing and preservingthese pharmaceutical compositions.

It has been discovered that clevidipine degrades under adverseconditions into several impurities that compromise the purity andultimately the potency of clevidipine. For example, under adverseconditions, clevidipine metabolizes into H168/79, which is also calledmethyl4-(2′,3′-dicloropenyl)-1,4-dihydro-2,6,-dimethyl-5-pyridine-carboxylate,and is shown in the following formula:

Based on this discovery, a degradation pathway of clevidipine isproposed, and is shown in FIG. 1. This pathway includes a number ofclevidipine degradation products, such as H324/78, H152/66, H152/81,H168/79, H207/59, and H207/36, for example. The pathway also illustratesthe further degradation of H168/79, by way of hydrolysis andcondensation, into Substance 23, Substance 24, and/or Substance 25. Thecomposition of Substance 23, Substance 24, and Substance 25 is asfollows:

Impurity levels were evaluated to determine the stability of clevidipineemulsions and their ability to minimize degradation of clevidipine underexposure to light. In addition H152/81, H168/79 and H207/59 aremetabolites of clevidipine. H324/78 is a pyridine analog of the activeingredient and is formed by the oxidation of clevidipine. Degradationproduct H152/81 is a dihydropyridine carboxylic acid and is formed fromthe hydrolysis of clevidipine. H152/81 can undergo decarboxylation andoxidation sequentially to form H168/79 and H207159 respectively. Thedegradation product H168/79 can undergo hydrolysis to form the diketoester H207/36 which can further cyclize to form substitutedcyclohexenone derivative impurities such as Substance 23 and Substance25. Substance 24, a diastereomer of Substance 23, may also form duringthis reaction.

As with any of the exemplary embodiments of pharmaceutical compositionsdescribed herein, it is preferred that the level of impurities in thecomposition is as low as possible. Therefore, while various exemplaryembodiments of pharmaceutical compositions include amounts of impuritieswithin acceptable and effective ranges for the compositions as a whole,the more pure the composition, meaning the higher the percent ofclevidipine or any of its acceptable salt forms, the better.

Substance 23 is an impurity generated through the degradation ofH168/79. The degradation of H168/79 is accelerated when exposed totemperatures in excess of 25° C. as compared to temperatures below 5° C.Therefore, the level of Substance 23 increases at higher temperatures.

Similarly, Substance 25 is an impurity generated through the degradationof H168/79 and may increase in quantity as H168/79 degrades.

Previously unidentified Substance 24 is also a degradation product ofH168/79. Given that Substance 24 is a diastereomer of Substance 23, aseparate HPLC method was developed to validate and quantify Substance24.

The present invention further includes a method of identifying andquantifying levels of Substance 24 in pharmaceutical samples havingclevidipine as an active ingredient. In one embodiment, the method ofdetecting Substance 24 in pharmaceutical samples having clevidipine asan active ingredient includes the step of isolating the individualchemical compounds making up the degradants or impurities found in theclevidipine degradation pathway. This can be accomplished by columnchromatography, such as high pressure liquid chromatography (“HPLC”),for example. The pharmaceutical sample having clevidipine as an activeingredient can introduced in small volume to the column and theresulting analysis of the eluent may illustrate the isolation andidentification of peaks representative of Substance 24. As may beunderstood by those skilled in the art, any optimization of the HPLCmethod may be performed to give the best separation of peaks as betweenthe various impurities found in the degradation of clevidipine.

Typical HPLC methods useful in the present invention are presented inExamples 1 and 2. Based on this method of detecting Substance 24, thelower limit of detection, or the minimum detectible level of Substance24, may be approximately 0.01% area of the pharmaceutical compositioncontaining clevidipine as an active ingredient. Alternatively, therecould be a lower limit of Substance 24, where the ratio of clevidipineto Substance 24 may be equal or similar to 9000 to 1 which is equal toor similar to 0.01%. Similarly, the lower limit of Substance 25 orSubstance 23 detection may be set forth as a ratio of clevidipine toSubstance 25 or Substance 23, where the ratio of clevidipine toSubstance 25 or Substance 23 may be equal or similar to 9000 to 1 whichis equal to or similar to 0.01%.

Example 1 HPLC Procedure

Clevidipine assay and related substances were tested at each time pointby a stability indicating method. This method is an isocratic, normalphase HPLC method with peak detection at 220 nm wavelength.

Column temperature: 35-40 degrees C.

Injection volume: 20 μl.

Flow rate: 1.0 ml/min.

Run time about 25 minutes.

Mobile phase of Heptane:ethanol (90:10) is employed and used for theassay of clevidipine and the degradation products with the exception ofSubstance 24.

Condition column with clevidipine mobile phase at 1.0 mL/min for 4hours.

New column should be conditioned overnight at 0.2 mL/min.

When a degradation product is eluted, column can be washed with filteredethanol for about 2 hours at 1.0 mL/min, then proceed withequilibration.

Examples of Column: PVA silica column 4.6 mm×150 mm, 5 micronPV12s051546WT or equivalent.

Example 2 HPLC Procedure Substance 24

This method is an isocratic, normal phase HPLC method with peakdetection at 220 nm wavelength.

Column temperature: 35-40 degrees C.

Injection volume: 20 μl to 100 μl.

Run time about 60 minutes.

Mobile phase of Heptane:Isopropyl Alcohol (95:5) is employed is used forthe assay of Substance 24.

Condition column with Heptane:Isopropyl Alcohol 95:5 mobile phase at 1.0ml/min until the blank injection baseline is stable. New column shouldbe conditioned overnight at 0.2 mL/min.

Examples of Column: Two PVA silica columns 4.6 mm×150 mm, 5 micronPV12s051546WT or equivalent.

Flow rate 1.0 mL/min.

Calculation of percent impurity based on total peak area:

$\begin{matrix}\frac{{impurity}\mspace{14mu}{Peak}\mspace{14mu}{{Area}(100)}}{\begin{matrix}\left( {{{total}\mspace{14mu}{peak}\mspace{14mu}{area}\mspace{14mu}{of}\mspace{14mu}{degradation}\mspace{14mu}{products}} +} \right. \\\left. {H\; 324\text{/}38\mspace{14mu}{peak}\mspace{14mu}{{area}\left( {{clevidipine}\mspace{14mu}{peak}\mspace{14mu}{area}} \right)}} \right)\end{matrix}} & {{Equation}\mspace{14mu}{\# 1}}\end{matrix}$

Calculation of percent impurity based on total peak area using

H168/79

as the impurity example:

$\begin{matrix}\frac{H\; 168\text{/}79\mspace{14mu}{Peak}\mspace{14mu}{{Area}(100)}}{\begin{matrix}\left( {{{total}\mspace{14mu}{peak}\mspace{14mu}{area}\mspace{14mu}{of}\mspace{14mu}{degradation}\mspace{14mu}{products}} +} \right. \\\left. {H\; 324\text{/}38\mspace{14mu}{peak}\mspace{14mu}{{area}\left( {{clevidipine}\mspace{14mu}{peak}\mspace{14mu}{area}} \right)}} \right)\end{matrix}} & {{Equation}\mspace{14mu}{\# 2}}\end{matrix}$

When a standard of a particular decomposition product is available,quantization of the impurity may be accomplished by standard proceduresknown in the art such as constructing a standard curve or by calculatinga relative response factor (RRF). When a standard is not available aratio of the area under the curve for the impurity to clevidipine can beused assuming a RRF previously calculated or if the RRF is not known anRRF of 1.0 is used to calculate the percent impurity

The present invention includes pharmaceutical compositions havingclevidipine as an active ingredient, wherein the level of impurityH168/79 is no more than 1.5% on a weight-by-weight basis. In oneembodiment of the present invention, the pharmaceutical compositionincludes clevidipine as an active ingredient and an amount of H168/79that is no greater than about 1.2%. In other embodiments, the amount ofH168/79 is preferably no greater than about 1.0%, and most preferably nogreater than about 0.5%. These compositions may further include otherdegradants in variable amounts as described herein, provided therequired level of potency of clevidipine remains satisfactory andeffective for use to treat any indication as described or incorporatedby reference herein.

The pharmaceutical composition exemplified in Tables 1, 2 and 3 areemulsions. The emulsions comprise: clevidipine 0.5 mg/ml., egg yolkphospholipid 1.2%, soybean oil 20%, glycerol 2.25%. The remainder beingwater adjusted to a pH between 6 and 8.8. The products were packaged in100 ml glass type II bottles with 28 mm West compound 1821 black stopperand aluminum seal.

In another embodiment of the present invention, the pharmaceuticalcomposition includes clevidipine as an active ingredient and H168/79,where the ratio of areas under the peak from an HPLC chromatogrambetween clevidipine and H168/79 is equal or above 60 to 0.9.Alternatively, the ratio between clevidipine and H168/79 can be equal toor above 100 to 1, 200 to 1, or 1000 to 1. In other embodiments, theratio between clevidipine and H168/79 can be between 2000 to 1 and 1000to 1. These compositions may further include other degradants invariable amounts as described herein, provided the required level ofpotency of clevidipine remains satisfactory and effective for use totreat any indication as described or incorporated by reference herein.

The percentage of H168/79 increases and stabilizes at lower temperaturesapproaching about 5° C., while it decreased at a temperaturesapproaching about 25° C. to about 40° C., for example. This trend showsthat a higher temperatures, H168/79 undergoes further degradation toSubstance 23, Substance 24, and/or Substance 25, by way of intermediaryH207/36, as illustrated in FIG. 1.

Thus, as illustrated in Table 1, Table 2 and Table 3 below, the loweringof temperature also provides stability for H168/79 and, as aconsequence, lowers and inhibits the amount of resulting second orderimpurities.

TABLE 1 Percentage of H168/79 Time KV 1322 KV 1328 KV 1329 TMC 001 TMC002  0 mo 0.4 0.3 0.3 0.2 0.3 5° C. upright 5° C. Inverted Time KV 1322KV 1328 KV 1329 TMC 001 TMC 002 Time KV 1322 KV 1328 KV 1329 TMC 001 TMC002  3 mo 0.1 <0.1 <0.1 0.1 0.1  3 mo 0.2 <0.1 <0.1 <0.1 0.2  6 mo 0.20.1 0.1 0.2 0.2  6 mo 0.2 0.1 0.1 0.2 0.2  9 mo 0.3 0.1 0.3 —

0.3  9 mo 0.3 0.1 0.3 0.2 0.3 12 mo 0.3 —

0.5 0.3 0.5 12 mo 0.3 —

0.4 0.3 0.5 18 mo 0.6 0.3 0.3 0.5 0.6 18 rno 0.6 0.3 0.3 0.4 0.6 24 mo0.6 0.3 0.3 0.5 0.7 24 mo 0.7 0.3 0.4 0.5 0.6 30 mo 0.6 0.4 0.3 0.5 0.730 mo 0.6 0.4 0.4 0.5 0.7 25° C. upright 25° C. inverted Time KV 1322 KV1328 KV 1329 TMC 001 TMC 002 Time KV 1322 KV 1328 KV 1329 TMC 001 TMC002  3 mo 0.8 0.6 0.4 0.7 1.0  3 mo 0.8 0.5 0.4 0.7 1.0  6 mo 0.7 0.40.4 0.5 0.9  6 mo 0.6 0.4 0.3 0.3 0.9  9 mo 0.5 0.4 0.4 0.5 —

 9 mo 0.5 0.4 0.4 —

0.8 12 mo 0.4 —

0.4 0.4 0.5 12 mo 0.4 —

0.4 0.4 0.5 18 mo 0.4 0.3 0.2 0.4 0.5 18 mo 0.4 0.3 0.2 0.5 0.5 24 mo0.3 0.3 0.2 0.3 0.4 24 mo 0.2 0.3 0.2 0.3 0.4 40° C. upright 40° C.inverted Time KV 1322 KV 1328 KV 1329 TMC 001 TMC 002 Time KV 1322 KV1328 KV 1329 TMC 001 TMC 002  1 mo 0.9 0.6 0.4 0.9 1.2  1 mo 0.0 0.6 0.40.9 1.3  2 mo 0.6 —

0.3 0.6 1.1  2 mo 0.7 —

0.3 0.7 1.0  3 mo 0.4 0.3 0.3 0.5 0.0  3 mo 0.4 0.3 0.3 0.5 0.7  6 mo0.3 0.2 0.2 0.6 0.4  6 mo 0.3 0.3 0.2 0.3 0.4

indicates data missing or illegible when filed

TABLE 2 Percentage of Substance 23 Time KV 1322 KV 1328 KV 1329 TMC 001TMC 002  0 mo ND ND ND ND ND 5° C. upright 5° C. Inverted Time KV 1322KV 1328 KV 1329 TMC 001 TMC 002 Time KV 1322 KV 1328 KV 1329 TMC 001 TMC002  3 mo ND ND ND ND ND  3 mo ND ND ND ND ND  6 mo 0.1 ND ND ND ND  6mo <0.1 ND ND ND ND  9 mo ND ND ND —

ND  9 mo ND ND ND ND ND 12 mo ND —

ND ND ND 12 mo ND —

ND ND ND 18 mo ND ND <0.1 ND ND 18 mo ND ND ND ND ND 24 mo <0.1 ND ND<0.1 <0.1 24 mo <0.1 ND <0.1 <0.1 <0.1 30 mo <0.1 <0.1 ND ND ND 30 mo ND<0.1 ND ND ND 25° C. upright 25° C. Inverted Time KV 1322 KV 1328 KV1329 TMC 001 TMC 002 Time KV 1322 KV 1328 KV 1329 TMC 001 TMC 002  3 moND ND ND <0.1 0.1  3 mo <0.1 ND ND <0.1 0.1  6 mo 0.2 <0.1 ND 0.2 0.3  6mo 0.2 ND ND 0.3 0.3  9 mo 0.3 0.1 ND 0.3 —

 9 mo 0.4 0.1 ND —

0.4 12 mo 0.4 —

0.1 0.4 0.6 12 mo 0.4 —

0.1 0.4 0.6 18 mo 0.4 0.2 0.5 0.6 0.6 18 mo 0.4 0.2 0.4 0.5 0.6 24 mo0.3 0.6 0.3 0.7 0.8 24 mo 0.3 0.5 0.6 0.8 0.9 40° C. upright 40° C.inverted Time KV 1322 KV 1328 KV 1329 TMC 001 TMC 002 Time KV 1322 KV1328 KV 1329 TMC 001 TMC 002  1 mo ND ND 0.1 0.1 0.1  1 mo ND ND 0.1 0.10.1  2 mo 0.2 —

0.2 0.3 0.4  2 mo 0.3 —

0.1 0.3 0.4  3 mo 0.4 0.3 0.2 0.4 0.5  3 mo 0.3 0.2 0.2 0.4 0.5  6 mo0.8 0.6 ND 1.1 1.1  6 mo 0.8 0.6 ND 0.9 1.0

indicates data missing or illegible when filed

TABLE 3 Percentage of Substance 25 Time KV 1322 KV 1328 KV 1329 TMC 001TMC 002  0 mo ND ND ND ND ND 5° C. upright 5° C. Inverted Time KV 1322KV 1328 KV 1329 TMC 001 TMC 002 Time KV 1322 KV 1328 KV 1329 TMC 001 TMC002  3 mo ND ND ND ND ND  3 mo ND ND ND ND ND  6 mo <0.1 ND ND ND 0.3  6mo <0.1 ND ND ND 0.2  9 mo ND <0.1 0.1 —

0.1  9 mo ND <0.1 <0.1 0.1 0.1 12 mo ND —

<0.1 0.1 ND 12 mo ND —

<0.1 0.1 ND 18 mo ND ND 0.1 ND ND 18 mo ND ND <0.1 ND ND 24 mo ND ND<0.1 ND <0.1 24 mo ND ND <0.1 ND <0.1 30 mo ND <0.1 ND ND ND 30 mo ND<0.1 ND ND ND 25° C. upright 25° C. Inverted Time KV 1322 KV 1328 KV1329 TMC 001 TMC 002 Time KV 1322 KV 1328 KV 1329 TMC 001 TMC 002  3 moND ND ND ND ND  3 mo ND ND ND ND ND  6 mo <0.1 ND ND <0.1 0.2  6 mo <0.1ND ND ND 0.2  9 mo 0.1 <0.1 0.1 0.1 —

 9 mo 0.2 <0.1 <0.1 —

0.2 12 mo 0.2 —

0.1 0.1 0.3 12 mo 0.2 —

0.1 0.1 0.3 18 mo 0.3 0.2 0.1 0.3 0.3 18 mo 0.2 0.2 <0.1 0.3 0.4 24 mo0.4 0.3 0.3 0.4 0.5 24 mo 0.3 0.3 0.3 0.4 0.5 40° C. upright 40° C.Inverted Time KV 1322 KV 1328 KV 1329 TMC 001 TMC 002 Time KV 1322 KV1328 KV 1329 TMC 001 TMC 002  1 mo ND ND <0.1 ND ND  1 mo ND ND <0.1 NDND  2 mo 0.1 —

<0.1 0.1 <0.1  2 mo 0.1

<0.1 0.1 0.2  3 mo 0.2 0.2 0.2 0.1 0.2  3 mo 0.2 0.2 0.2 0.1 0.2  6 moND 0.3 0.3 0.6 0.7  6 mo <0.1 0.3 0.3 ND 0.6

indicates data missing or illegible when filed

According to an aspect of the present invention, the above mentionedmethods of stabilizing pharmaceutical compounds having clevidipine as anactive ingredient provide a shelf life of at least 36 months for thecompositions, when stored at about 2° C. to 8° C. After being removedfrom this refrigerated condition and placed at roughly room temperature(15° C. to 30° C.), the compositions remain stable for up to at least 2additional months.

The present invention also includes a method of maintaining thestability of a pharmaceutical composition having clevidipine as theactive ingredient, including the slowing down or otherwise inhibiting ofthe hydrolysis pathway of clevidipine by reducing or inhibiting theamount of water in the process of manufacturing the composition, as wellas the emulsification process in the final formulation.

In one embodiment of the present invention, clevidipine is manufacturedby reaction of4-(2′,3′-dichlorophenyl)-1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3-pyridinecarboxylicacid with chloromethyl butyrate to obtain clevidipine. This reaction canbe done optionally in the presence of a corresponding hydrogencarbonate, such as KHCO₃, in refluxing acetonitrile. Inorganic salts canbe removed by filtration and the product is crystallized by the additionof isopropanol with subsequent cooling. It can also be crystallized byexchanging solvent from acetonitrile to a mixture of alcohol, such asethanol or isopropanol, with repeated evaporations. In the furtherpurification of the product the crystals are washed with a mixture ofethanol or isopropanol. The product can be dissolved in refluxingisopropanol, crystallized by cooling, isolated by filtration and finallywashed with an isopropanol mixture. A more detailed description of themanufacturing process of clevidipine can be found in U.S. Pat. No.6,350,877, the entire disclosure of which is incorporated by referenceherein as if set forth in its entirety.

Clevidipine is typically formulated as a liquid emulsion suitable forintravenous administration. Lipid emulsions are widely used inparenteral nutrition use for approximately 30 years and in the recentpast have been used as drug carriers for insoluble drugs such aspropofol (Diprivan®), and diazepam. Apart from their ability to deliverinsoluble drugs, emulsions are also suitable dosage forms for drugs likeclevidipine that are susceptible to hydrolytic breakdown. Emulsions havealso been reported to prevent drugs from adhering to plasticadministration sets used during intravenous injection, and reduce localtoxicity on infusion.

Typically, each mL may contain 0.5 mg clevidipine in approximately 20%soybean oil emulsion for intravenous administration. Other ingredientsmay include glycerin, purified egg yolk phospholipids and sodiumhydroxide to adjust pH. Generally, water for injection is dispensed to amix tank at about 74° C. to about 78° C. Glycerin is added, and theaqueous phase is cooled to about 60° C. to about 70° C. prior toaddition of the oil phase. For the oil phase, soybean oil is dispensedinto a dissolving tank, mixed and heated to about 70° C. to about 82° C.Clevidipine is then added to the soybean oil mixture and heated to about78° C. to about 82° C. Egg yolk phospholipids are then added to themixture. The aqueous and oil phases are mixed together to form anemulsion, and the pH is adjusted with 1N sodium hydroxide to a pH ofabout 6 to about 8.8. The emulsion is then homogenized at a pressure ofabout 500 to 8000 psi and a temperature of about 50° C. to about 55° C.to a fine particle size. Preferably, the emulsion is homogenized atabout 25° C., More preferably at about 15° C., still more preferably atabout 10° C. and most preferably at about 5° C. The samples are filteredand dispensed into 50 mL or 100 mL bottles and capped with siliconizedrubber stoppers, and crimp sealed with an aluminum overseal. Furtherinformation regarding the formulation of clevidipine can be found inU.S. Pat. No. 5,739,152, the entire disclosure of which is incorporatedby reference herein as if set forth in its entirety.

Emulsions of the present invention comprise an oil-in-water emulsioncomprising: a) clevidipine, b) a lipid phase, c) an emulsifier, and d)water or a buffer. The emulsion may also contain co-solvents or othersolubility enhancers, antioxidants, stabilizers, pH-adjusting agents ortonicity modifying agents, such as glycerol.

In an emulsion of the present invention, clevidipine is present fromabout 0.4 mg/ml to about 0.6 mg/ml, Preferably clevidipine is presentfrom about 0.45 mg/ml to about 0.55 mg/ml. The lipid phase is presentfrom about 1% to about 35%, preferably from about 18% to about 22%. Theemulsifier is present from about 0.01 to about 2 times the weight of thelipid phase, preferably from about 0.5% to about 4% and more preferablyfrom about 1% to about 1.32%. The remainder of the emulsion is water orbuffer. The preferred range of water or buffer is about 75% to about90%. The pH of the emulsion is adjusted to about 6 to about 8.8,preferably from about 7.5 to about 8.8. When present, glycerol ispresent from about 2% to about 2.5%. Percentages of the emulsioncomposition are expressed as weight/weight.

Lipid phases in the emulsion are any pharmaceutically acceptable oil,preferably triglycerides such as soy bean oil, safflower seed oil, oliveoil, cottonseed oil, sunflower oil, sesame oil, peanut oil, corn oil,medium chain triglycerides (such as Miglyol® 812 or 810) or triacetin.The lipid phase may also be propylene glycol diesters or monoglycerides(such as acetylareal monoglycerides). The lipid phase can also be amixture of said ingredients. The most preferred lipid phase is soy beanoil.

Emulsifiers are any pharmaceutically acceptable emulsifier, preferablyphospholipids extracted from egg yolk or soy bean, syntheticphosphatidyl cholines or purified phosphatidyl cholines from vegetableorigin. Hydrogenated derivatives can also be used, such as phosphatidylcholine hydrogenated (egg) and phosphatidyl choline hydrogenated (soya).Emulsifiers may also be non-ionic surfactants such as poloxamers (forexample Poloxamer 188 and 407), poloxamines, polyoxyethylene stearates,polyoxyethylene sorbitan fatty acid esters or sorbitan fatty acidesters. Ionic suffactants may also be used such as cholic acid anddeoxycholic acid or surface active derivatives or salts thereof. Theemulsifier can also be a mixture of said ingredients. The most preferredemulsifier is egg yolk phospholipid.

Those of ordinary skill in the art will recognize that manymodifications and variations of the present invention may be implementedwithout departing from the spirit or scope of the invention. Thus, it isintended that the present invention cover the modification andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

1. A pharmaceutical composition for treating or preventing hypertensionin a subject in need thereof, comprising clevidipine or any of itspharmaceutically acceptable salts as an active ingredient, wherein thecomposition is stored at an effective temperature so that the level of adegradant is approximately equal to or less than 0.2% on aweight-by-weight basis, wherein the degradant is selected from a grouphaving the formula:


2. The composition of claim 1, wherein the effective temperature isapproximately equal to or less than about 25° C.
 3. The composition ofclaim 1, wherein the effective temperature is approximately equal to orless than about 5° C.
 4. A pharmaceutical composition for treating orpreventing hypertension in a subject in need thereof, comprisingclevidipine or any of its pharmaceutically acceptable salt as an activeingredient, wherein the composition is stored at an effectivetemperature so that the level of a degradant, H168/79, is approximatelyequal to or less than 1.5% on a weight-by-weight basis, wherein H168179having the formula:


5. The composition of claim 4, wherein the effective temperature isapproximately equal to or less than about 25° C.
 6. The composition ofclaim 4, wherein the effective temperature is approximately equal to orless than about 5° C.
 7. A pharmaceutical composition comprisingclevidipine and a degradant having the formula:

prepared by a process comprising: storing the pharmaceutical compositionat an effective temperature wherein the amount of degradant isapproximately equal to or less than about 0.2%.
 8. The composition ofclaim 7, wherein the effective temperature is approximately equal to orless than about 25° C.
 9. The composition of claim 7, wherein theeffective temperature is approximately equal to or less than about 5° C.10. A pharmaceutical composition comprising an effective amount ofclevidipine and Substance 23, wherein the ratio of the HPLC peak areasbetween clevidipine and Substance 23 is equal or above 500 to
 1. 11. Apharmaceutical composition comprising an effective amount of clevidipineand Substance 24, wherein the ratio of the HPLC peak areas betweenclevidipine and Substance 24 is equal or above 500 to
 1. 12. Apharmaceutical composition comprising an effective amount of clevidipineand Substance 25, wherein the ratio of the HPLC peak areas betweenclevidipine and Substance 23 is equal or above 500 to
 1. 13. Apharmaceutical composition comprising clevidipine and a degradant havingthe following formula:

wherein the amount of degradant is approximately equal to or less thanabout 1.5% on a weight by weight basis.
 14. The composition of claim 13,wherein the amount of degradant is approximately equal to or less thanabout 1.0%.
 15. The composition of claim 13, wherein the amount ofdegradant is approximately equal to or less than about 0.5%.
 16. Amethod of reducing impurities in a composition, comprising: heating anoil to between to about 70° C. to about 82° C.; adding to the heated oilclevidipine and heating the mixture to about 78° C.; adding to themixture egg yolk phospholipids; and adding an aqueous phase; wherein thepH is adjusted to about 6 to about 8.8; homogenizing the emulsion; andwherein the amount of degradant with the formula of:

is approximately equal to or less than about 1.5%.
 17. The compositionof claim 16, wherein the amount of degradant is approximately equal toor less than about 1.0%.
 18. The composition of claim 16, wherein theamount of degradant is approximately equal to or less than about 0.5%.19. The composition of claim 16, wherein the aqueous phase consistsessentially of glycerin.
 20. The method of claim 16, wherein the pH isabout 9.8.
 21. The method of claim 16, wherein the mixture ishomogenized at about 25° C.
 22. The method of claim 16, wherein themixture is homogenized at about 15° C.
 23. The method of claim 16,wherein the mixture is homogenized at about 10° C.
 24. The method ofclaim 16, wherein the mixture is homogenized at about 5° C.
 25. Apharmaceutical composition comprising an effective amount of clevidipineprepared by a process comprising: heating an oil to between to about 70°C.; adding to the heated oil clevidipine and heating the mixture toabout 78° C.; adding to the mixture egg yolk phospholipids; and addingan aqueous phase; wherein the aqueous phase consists essentially ofglycerin and the pH is adjusted to about 9.5; and wherein the amount ofdegradant with the formula of:

is approximately equal to or less than about 1.5%.
 26. The compositionof claim 25, wherein the amount of degradant is approximately equal toor less than about 1.0%.
 27. The composition of claim 25, wherein theamount of degradant is approximately equal to or less than about 0.5%.28. Method of detecting Substance 24, comprising optimizing an HPLCmethod to detect Substance 24 wherein the ratio of the HPLC peak areasbetween clevidipine and Substance 24 is equal or above 500 to 1.